Compositions and methods for inhibiting pathogenic growth

ABSTRACT

The invention includes methods and compositions for treating an animal to inhibit the incidence and growth of  E. coli  O157:H7 and other pathogenic bacteria. The treatment method comprises administering a therapeutically effective amount of  Lactobacillus acidophilus  or one or a combination of a number of other probiotic bacteria to an animal. An alternative treatment method comprises administering a therapeutically effective amount of a lactic acid producing bacterium such as  Lactobacillus acidophilus  in combination with a lactate utilizing bacterium such as  Propionibacterium freudenreichii.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/905,215filed Dec. 21, 2004, now abandoned, which is a divisional of U.S.application Ser. No. 10/288,487, filed Nov. 6, 2002, now U.S. Pat. No.7,063,836, which is a continuation of U.S. application Ser. No.10/273,141, filed Oct. 18, 2002 (Abandoned); U.S. application Ser. No.10/288,487, filed Nov. 6, 2002, now U.S. Pat. No. 7,063,836, also claimspriority to U.S. Provisional Application No. 60/319,054, filed Jan. 8,2002 (Expired), and U.S. Provisional Application No. 60/319,587, filedOct. 1, 2002 (Expired). This application is also a continuation of U.S.application Ser. No. 11/160,470, filed Jun. 24, 2005, now abandoned,which is a continuation of U.S. application Ser. No. 10/288,487, filedNov. 6, 2002, now U.S. Pat. No. 7,063,836, which is a continuation ofU.S. application Ser. No. 10/273,141, filed Oct. 18, 2002 (Abandoned);U.S. application Ser. No. 10/288,487, filed Nov. 6, 2002, now U.S. Pat.No. 7,063,836, also claims priority to U.S. Provisional Application No.60/319,054, filed Jan. 8, 2002 (Expired), and U.S. ProvisionalApplication No. 60/319,587, filed Oct. 1, 2002 (Expired). All of theseapplications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for inhibitingpathogenic growth. More specifically, the invention relates tocompositions and methods for inhibiting pathogenic growth through theuse of lactic acid producing microorganisms both alone and incombination with lactate utilizing microorganisms.

BACKGROUND OF THE INVENTION

Ingestion of pathogens, especially bacterial pathogens, but includingviruses and other disease causing microorganisms, is a common problem inmost animals. Pathogens have been known to cause illnesses in animalsthat have wide ranging deleterious effects including weight loss,diarrhea, abdominal cramping, and renal failure. For animals that areimmunosuppressed or malnourished, even just the effects of diarrhea canbe fatal. Pathogens are often transferred between animals where poorhygiene conditions exist, and sometimes communicability cannot beprevented even when great care is taken. The most common solution tothis problem has been to provide antibiotics to the animals; however,this solution is not only costly, but it also can result in thegeneration of antibiotic-resistant strains of bacteria.

Extreme health risks result when humans consume pathogens incontaminated food products such as sprouts, lettuce, meat products,unpasteurized milk and juice, and sewage-contaminated water, forexample. The problem is particularly prevalent in the beef and dairyindustry. Pathogens present on a cow's udder or on milking equipment mayfind their way into raw milk. Meat can become contaminated duringslaughter, and pathogenic organisms can be mixed into large quantitiesof meat when it is ground. When humans eat meat, especially ground beef,that has not been cooked sufficiently to kill any pathogens present inthe beef, serious and life-threatening infections can result. This is adifficult problem to solve because contaminated meat often looks andsmells perfectly normal. Furthermore, the number of pathogenic organismsneeded to cause disease is extremely small, thus making detectionextraordinarily difficult.

Pathogens that cause disease in the intestinal tract are known asenteropathogens. Examples of enteropathogenic bacteria, orenterobacteria, include Staphylococcus aureus, particular strains ofEscherichia coli (E. coli), and Salmonella spp. Whereas most of thehundreds of strains of E. coli are harmless and live in the intestinesof animals, including humans, some strains, such as E. coli O157:H7,O111:H8, and O104:H21, produce large quantities of powerful shiga-liketoxins that are closely related to or identical to the toxin produced byShigella dysenteriae. These toxins can cause severe distress in thesmall intestine, often resulting in damage to the intestinal lining andresulting in extreme cases of diarrhea. E. coli O157:H7 can also causeacute hemorrhagic colitis, characterized by severe abdominal crampingand abdominal bleeding. In children, this can progress into the rare butfatal disorder called hemolytic uremic syndrome (“HUS”), characterizedby renal failure and hemolytic anemia. In adults, it can progress intoan ailment termed thrombotic thrombocytopenic purpura (“TTP”), whichincludes HUS plus fever and neurological symptoms and can have amortality rate as high as fifty percent in the elderly.

Reduction of risk for illnesses due to food borne pathogens can beachieved by controlling points of potential contamination. The beefindustry has recognized the need to investigate pre-harvest control ofpathogens, particularly E. coli O157:H7, due to potential runoffcontamination, contact with humans, and the transfer of pathogens duringmeat processing. In particular, undercooked or raw hamburger (groundbeef) has been implicated in many documented outbreaks as containing E.coli O157:H7.

Accordingly, there is a recognized need for compositions and methods forreducing or eliminating the growth of enteropathogens such as E. coliO157:H7 for the health benefits to the animals. Furthermore, there is animportant need for reducing or eliminating the growth of enteropathogensin meat and milk producing animals prior to their harvest for thebenefit of consumers. By such reduction or elimination in food animals,consumers of beef, dairy, and other food products will be betterprotected from the risk of consuming such pathogens.

SUMMARY OF THE INVENTION

Since pathogens are known to populate many distinct areas of animals'digestive tracts, it has been found to be most beneficial to supply andpotentiate those organisms that occur naturally in those areas and whichare effective for inhibiting pathogenic growth throughout the digestivetract, such as the rumen, small intestine, and large intestine. Thepresent invention identifies such naturally occurring organisms suitablefor serving this purpose and demonstrates methods for enhancing theirpopulations and efficacy. The microorganisms in the formulations andmethods of the present inventions may individually and collectivelyproduce compounds that inhibit the growth of pathogens in thegastrointestinal tract (“GIT”) of animals. By inhibiting the growth ofthe pathogens, the methods and compounds of the invention provide areduced likelihood of contaminated food products resulting from treatedanimals.

The invention exploits the natural competition of certain microorganismswith the pathogenic organisms that it is the object of the invention toreduce or destroy. The microorganisms in the formulations of theinvention may exhibit multifaceted modes of action. These actions rangefrom complex actions such as acting as or producing bactericides tosimply competing with the pathogen by using more nutrients andattachment spaces than the pathogens, thus preventing them from becomingestablished within the GIT. These advantageous action modes can becontrasted with less advantageous techniques conventionally known forachieving such effects as using aseptic husbandry accompanied by theaddition of antibiotics and like substances to animals' feed.

In the competitive mode of action, particularly of Lactobacillusacidophilus, including strain 381-IL-28 (also known as and referred tothroughout as the LA51 strain and NPC747), the microorganisms out-growand out-populate E. coli O157:H7, thereby acting as an inhibitor to thatpathogen. E. coli O157:H7 and Lactobacillus acidophilus are understoodto at least partly utilize the same limited supply of in vitro nutrientssuch as sugar. Furthermore, these microorganisms compete for the sameattachment space: on the lining of the GIT. With a rapid-proliferationinhibitor such as Lactobacillus acidophilus, the primary mode of actionagainst E. coli O157:H7 is to overwhelm it by using the available foodand suitable attachment spaces.

The invention includes a method of treating or preventing an intestinalpathogenic infection in a ruminant comprising administering to theruminant a composition comprising a therapeutically effective amount ofa lactic acid producing bacterium, wherein the lactic acid producingbacterium reduces the quantity of a pathogen in the intestine of theruminant. In one embodiment, the lactic acid producing bacterium isselected from the group consisting of: Bacillus subtilis,Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacteriumbifudum, Bifidobacterium infantis, Bifidobacterium longum,Bifidobacterium thermophilum, Lactobacillus acidophilus, Lactobacillusagilis, Lactobacillus alactosus, Lactobacillus alimentarius,Lactobacillus amylophilus, Lactobacillus amylovorans, Lactobacillusamylovorus, Lactobacillus animalis, Lactobacillus batatas, Lactobacillusbavaricus, Lactobacillus bifermentans, Lactobacillus bifidus,Lactobacillus brevis, Lactobacillus buchnerii, Lactobacillus bulgaricus,Lactobacillus catenaforme, Lactobacillus casei, Lactobacilluscellobiosus, Lactobacillus collinoides, Lactobacillus confusus,Lactobacillus coprophilus, Lactobacillus coryniformis, Lactobacilluscorynoides, Lactobacillus crispatus, Lactobacillus curvatus,Lactobacillus delbrueckii, Lactobacillus desidiosus, Lactobacillusdivergens, Lactobacillus enterii, Lactobacillus farciminis,Lactobacillus fermentum, Lactobacillus frigidus, Lactobacillusfructivorans, Lactobacillus fructosus, Lactobacillus gasseri,Lactobacillus halotolerans, Lactobacillus helveticus, Lactobacillusheterohiochii, Lactobacillus hilgardii, Lactobacillus hordniae,Lactobacillus inulinus, Lactobacillus jensenii, Lactobacillus jugurti,Lactobacillus kandleri, Lactobacillus kefir, Lactobacillus lactis,Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillusmalefermentans, Lactobacillus mali, Lactobacillus maltaromicus,Lactobacillus minor, Lactobacillus minutus, Lactobacillus mobilis,Lactobacillus murinus, Lactobacillus pentosus, Lactobacillus plantarum,Lactobacillus pseudoplantarum, Lactobacillus reuteri, Lactobacillusrhamnosus, Lactobacillus rogosae, Lactobacillus tolerans, Lactobacillustorquens, Lactobacillus ruminis, Lactobacillus sake, Lactobacillussalivarius, Lactobacillus sanfrancisco, Lactobacillus sharpeae,Lactobacillus trichodes, Lactobacillus vaccinostercus, Lactobacillusviridescens, Lactobacillus vitulinus, Lactobacillus xylosus,Lactobacillus yamanashiensis, Lactobacillus zeae, Pediococcusacidlactici, Pediococcus pentosaceus, Streptococcus cremoris,Streptococcus discetylactis, Streptococcus faecium, Streptococcusintermedius, Streptococcus lactis, Streptococcus thermophilus, andcombinations thereof. In one embodiment, the lactic acid producingbacterium is Lactobacillus acidophilus. In another embodiment, theLactobacillus acidophilus strains include the M35, LA45, LA51 and L411strains. In another embodiment, the Lactobacillus acidophilus strain isLA51. The lactic acid producing bacterium may be administered at a levelof at least 1×10⁸ CFU/day. Alternatively, the lactic acid producingbacterium may be administered at a level of about 1×10⁹ CFU/day. Thepathogen may be selected from the group consisting of E. coli,Salmonella spp., including Salmonella typhirium, and Staphylococcusaureus. Alternatively, the pathogen may be E. coli O157:H7.

Another aspect of the invention includes a composition for treating orpreventing a pathogenic infection in a ruminant comprising aLactobacillus acidophilus strain selected from the group consisting ofM35, LA45, LA51 and L411 in combination with animal feed. In oneembodiment, the Lactobacillus acidophilus strain is LA45 or LA51. Inanother embodiment, the Lactobacillus acidophilus strain is LA51. TheLactobacillus acidophilus may be present in the animal feed in an amountof greater than 1×10⁸ CFU for each quantity of food equal to the amounteaten by one animal in one day, or the Lactobacillus acidophilus may bepresent in the animal feed in an amount of about 1×10⁹ CFU for eachquantity of food equal to the amount eaten by one animal in one day.

As already mentioned, strain LAM is also known as 381-IL-28, and isavailable under that accession number from the Oklahoma State Universitycollection. While the inventors have characterized LA51 as aLactobacillus acidophilus, other means of characterization haveidentified it as Lactobacillus animalis, and Lactobacillus murinus. LA45is deposited at the American Type Culture Collection under accessionnumber ATCC 53545. M35 and L411 are the accession numbers for thosebacteria available from the University of Nebraska. In addition, strainsC28, M35, LA45, and LA51 were deposited with the American Type CultureCollection (ATCC, Manassas, Va. 20110-2209) on May 25, 2005 and have theDeposit numbers of PTA-6748, PTA-6751, PTA-6749, and PTA-6750,respectively. Strain L411 was deposited with the American Type CultureCollection (ATCC) on Jun. 30, 2005 and have the Deposit number ofPTA-6820. These deposits were made in compliance with the BudapestTreaty requirements that the duration of the deposit should be forthirty (30) years from the date of deposit or for five (5) years afterthe last request for the deposit at the depository, or for theenforceable life of a patent that results from this application,whichever is longer. The strains will be replenished should it becomenon-viable at the Depository.

Another aspect of the invention includes a method of treating orpreventing an intestinal pathogenic infection in a ruminant, the methodcomprising administering to the ruminant a composition comprising atherapeutically effective amount of a lactic acid producing bacteriumand a lactate utilizing bacterium, wherein the lactic acid producingbacterium reduces the quantity of a pathogen in the intestine of theruminant. The lactic acid producing bacterium may be selected from thegroup consisting of: Bacillus subtilis, Bifidobacterium adolescentis,Bifidobacterium animalis, Bifidobacterium bifudum, Bifidobacteriuminfantis, Bifidobacterium longum, Bifidobacterium thermophilum,Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillusalactosus, Lactobacillus alimentarius, Lactobacillus amylophilus,Lactobacillus amylovorans, Lactobacillus amylovorus, Lactobacillusanimalis, Lactobacillus batatas, Lactobacillus bavaricus, Lactobacillusbifermentans, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillusbuchnerii, Lactobacillus bulgaricus, Lactobacillus catenaforme,Lactobacillus casei, Lactobacillus cellobiosus, Lactobacilluscollinoides, Lactobacillus confusus, Lactobacillus coprophilus,Lactobacillus coryniformis, Lactobacillus corynoides, Lactobacilluscrispatus, Lactobacillus curvatus, Lactobacillus delbrueckii,Lactobacillus desidiosus, Lactobacillus divergens, Lactobacillusenterii, Lactobacillus farciminis, Lactobacillus fermentum,Lactobacillus frigidus, Lactobacillus fructivorans, Lactobacillusfructosus, Lactobacillus gasseri, Lactobacillus halotolerans,Lactobacillus helveticus, Lactobacillus heterohiochii, Lactobacillushilgardii, Lactobacillus hordniae, Lactobacillus inulinus, Lactobacillusjensenii, Lactobacillus jugurti, Lactobacillus kandleri, Lactobacilluskefir, Lactobacillus lactis, Lactobacillus leichmannii, Lactobacilluslindneri, Lactobacillus malefermentans, Lactobacillus mali,Lactobacillus maltaromicus, Lactobacillus minor, Lactobacillus minutus,Lactobacillus mobilis, Lactobacillus murinus, Lactobacillus pentosus,Lactobacillus plantarum, Lactobacillus pseudoplantarum, Lactobacillusreuteri, Lactobacillus rhamnosus, Lactobacillus rogosae, Lactobacillustolerans, Lactobacillus torquens, Lactobacillus ruminis, Lactobacillussake, Lactobacillus salivarius, Lactobacillus sanfrancisco,Lactobacillus sharpeae, Lactobacillus trichodes, Lactobacillusvaccinostercus, Lactobacillus viridescens, Lactobacillus vitulinus,Lactobacillus xylosus, Lactobacillus yamanashiensis, Lactobacillus zeae,Pediococcus acidlactici, Pediococcus pentosaceus, Streptococcuscremoris, Streptococcus discetylactis, Streptococcus faecium,Streptococcus intermedius, Streptococcus lactis, Streptococcusthermophilus, and combinations thereof. The lactate utilizing bacteriummay be selected from the group consisting of Megasphaerae eilsdenii,Peptostreptococcus asaccharolyticus, Propionibacterium freudenreichii,Propionibacterium acid-propionici, Propionibacterium freudenreichii,Propionibacterium globosum, Propionibacterium jensenii,Propionibacterium shermanii, Propionibacterium spp., Selenomonasruminantium, and combinations thereof. In one embodiment, the lacticacid producing bacterium is Lactobacillus acidophilus. In anotherembodiment, the Lactobacillus acidophilus strain is selected from thegroup consisting of M35, LA45, LA51 and L411. In another embodiment, theLactobacillus acidophilus is the LA51 strain. In one embodiment, thelactate utilizing bacterium is Propionibacterium freudenreichii. Inanother embodiment, the Propionibacterium freudenreichii strain isselected from the group consisting of P9, PF24, P42, P93 and P99. Inanother embodiment, the Propionibacterium freudenreichii strain is PF24,available from the ATCC under accession number ATCC 9615. The lactateutilizing bacterium and the lactic acid producing bacterium may each beadministered in an amount of greater than 1×10⁸ CFU/day, or in an amountof about 1×10⁹ CFU/day. Alternatively, the lactate utilizing bacteriummay be administered in an amount of about 1×10⁶ CFU/day. In anotherembodiment the lactate utilizing bacterium is administered in an amountof greater than 1×10⁶ CFU/day, preferably in an amount of greater than1×10⁸ CFU/day, and most preferably in an amount of about 1×10⁹ CFU/day.Propionibacterium strain PF24 was also deposited with the American TypeCulture Collection (ATCC, Manassas, Va. 20110-2209) (ATCC) on May 25,2005 and has the Deposit number of PTA-6752. Strains P9 and P42 weredeposited with the ATCC on Jun. 30, 2005 and have the Deposit numbers ofPTA-6821 and PTA-6822, respectively. These deposits were made incompliance with the Budapest Treaty requirements that the duration ofthe deposit should be for thirty (30) years from the date of deposit orfor five (5) years after the last request for the deposit at thedepository, or for the enforceable life of a patent that results fromthis application, whichever is longer. The strains will be replenishedshould it become non-viable at the Depository.

Another aspect of the invention includes a composition for treating orpreventing a pathogenic infection in a ruminant comprising aLactobacillus acidophilus strain selected from the group consisting ofM35, LA45, LA51, L411, and combinations thereof, in combination with aPropionibacterium freudenreichii strain selected from the groupconsisting of P9, PF24, P42, P93, P99, and combinations thereof. In oneembodiment, the composition further comprises animal feed. In anotherembodiment, the Lactobacillus acidophilus and the Propionibacteriumfreudenreichii are each present in the animal feed in an amount ofgreater than 1×10⁸ CFU for each quantity of food equal to the amounteaten by one animal in one day. In another embodiment, the Lactobacillusacidophilus strain LA51 and Propionibacterium freudenreichii strain PF24are each present in the animal feed in an amount of about 1×10⁹ CFU foreach quantity of food equal to the amount eaten by one animal in oneday. In another embodiment, the composition further comprisesLactobacillus acidophilus strain LA45 present in the animal feed in anamount of about 1×10⁶ CFU for each quantity of food equal to the amounteaten by one animal in one day.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for reducing oreliminating the growth of pathogens in the gut of an animal. In vitroand in vivo tests have been conducted utilizing certain strains ofmicroorganisms, which have been found to be particularly effective atinhibiting the growth of many pathogens, including E. coli O157:H7. Asused herein, the term “pathogens” refers to any bacterium that producesa harmful effect in a host animal, and especially those bacteria thatinfect meat and dairy animals and subsequently infect the human foodsupply, thus causing disease in humans. The invention is considered tobe useful in preventing the growth of a wide variety of pathogenicorganisms, as demonstrated herein by several tests showing theinhibition of growth of pathogenic bacteria including E. coli,Salmonella spp., including Salmonella typhirium, and Staphylococcusaureus.

The formulations and methods described herein are applicable to a widevariety of animal species and commercial practices. The inhibition ofpathogens in the GIT of animals may be considered for those used in thecommercial production of meat, milk, poultry, and fish. In one aspect,the invention includes a method for treating an animal to inhibit theincidence and growth of E. coli O157:H7. The treatment method includesadministering a therapeutically effective amount of a selectedLactobacillus acidophilus to an animal that inhibits in vivo growth ofE. coli O157:H7. As used herein, the term “therapeutically effectiveamount” refers to the quantity of bacteria administered to an animalthat results in a therapeutic effect by creating an inhospitableenvironment for pathogens. It has been found that a therapeuticallyeffective amount of Lactobacillus acidophilus can be as little as 1×10⁶CFU/day when it is administered in combination with other components,although it is preferable that the lactic acid producing bacteria of theinvention are administered in an amount of greater than 1×10⁸ CFU/day.It has been found to be particularly effective when the selectedLactobacillus acidophilus is administered at a level of approximately1×10⁹ CFU/day.

Among the Lactobacillus acidophilus strains found to be particularlyeffective as E. coli O157:H7 inhibitors is the 381-IL-28, or the LA51strain. In one aspect, the invention includes Lactobacillus acidophilusstrains that are effective compositions in the above-described methodswhen provided as a product in the prescribed concentrations for animalconsumption as E. coli O157:H7 inhibitors. Before the present invention,Lactobacillus acidophilus microorganisms had been administered as animalfeed additives for different purposes such as better utilization offeed-stuffs. For example, U.S. Pat. Nos. 5,534,271 and 5,529,793(incorporated herein by reference), report that certain combinations oflactic acid producing bacteria and lactate utilizing bacteria could beused in a method to improve the utilization of feedstuffs by ruminants.The present invention, by contrast, sets forth methods for inhibitingpathogenic growth in animals and for improving the quality and quantityof dairy products. However, one aspect of the present invention includesthe discovery that certain novel formulations for inhibiting pathogenicgrowth disclosed herein are also useful for improving the utilization offeedstuffs. To the extent that these formulations were previouslyunknown for improving utilization of feedstuffs, they form part of thepresent invention for that purpose.

In one embodiment, the present invention includes a method for providinga product as an inhibitor of E. coli O157:H7 growth in animals. Themethod includes selecting a therapeutically effective microorganism asan E. coli O157:H7 inhibitor in animals and producing a productcontaining this microorganism. Generally, such products requiregovernment approval to be certified as pathogen inhibitors;specifically, certification from the United States Department ofAgriculture (USDA) is typically required. If the product is for humanconsumption, for example to counteract an E. coli infection in a human,approval by the United States Food and Drug Administration (FDA) isrequired.

An example of a microorganism found to be therapeutically effective isLactobacillus acidophilus, preferably the LA51 strain, which inhibits invivo growth of E. coli O157:H7 and other pathogenic microorganisms whenadministered to animals at a dose of approximately 1×10⁹ CFU/day.Alternatively, a sufficient level may be considered to be at least asmuch as 1×10⁸ CFU/day. Exact dosage levels can easily be determined bythose skilled in the art by evaluating the bile tolerance of thebacteria to be administered in order to verify that viable organisms aredelivered to the intestinal tract to compete with and inhibit the growthof pathogenic bacteria such as E. coli O157:H7.

The present invention identifies several naturally occurring organismsthat are capable of inhibiting pathogen growth within the GIT of ananimal. Since many pathogens are acid resistant and populate manydistinct areas of an animal's digestive tract, the naturally occurringorganisms of the invention are preferably capable of inhibiting pathogengrowth at a lower pH and in several areas of the GIT; e.g., the rumen,small intestine and large intestine. Earlier research has shown that E.coli O157:H7 populations may be decreased in cattle by feeding hayrations, which in and of itself increases rumen pH to 7.0. However, thishas limited application in the finishing or feedlot industries sinceanimals in this phase of the production process are typically fed a dietthat has a greater proportion of grain in order to foster better carcasscharacteristics.

Microorganisms that are useful in the formulations and methods of thepresent invention may be capable of producing lactic acid in the GIT.These microorganisms include, for example, the genera Lactobacillus orEnterococcus. Either or both genera may be used. They are distinguishedby their ability to utilize sugars such as glucose or lactose or, in thecase of Enterococcus, to utilize starch, to produce lactic acid, andthus reduce the local pH level. The choice of microorganism can dependupon the locus at which the desired effect is to be given. For example,the genus Lactobacillus is capable of reducing local pH more thanEnterococcus microorganisms.

Lactic acid producing organisms that may be used in the methods andcompositions of the invention include but are not limited to: Bacillussubtilis, Bifidobacterium adolescentis, Bifidobacterium animalis,Bifidobacterium bifudum, Bifidobacterium infantis, Bifidobacteriumlongum, Bifidobacterium thermophilum, Lactobacillus acidophilus,Lactobacillus agilis, Lactobacillus alactosus, Lactobacillusalimentarius, Lactobacillus amylophilus, Lactobacillus amylovorans,Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus batatas,Lactobacillus bavaricus, Lactobacillus bifermentans, Lactobacillusbifidus, Lactobacillus brevis, Lactobacillus buchnerii, Lactobacillusbulgaricus, Lactobacillus catenaforme, Lactobacillus casei,Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillusconfusus, Lactobacillus coprophilus, Lactobacillus coryniformis,Lactobacillus corynoides, Lactobacillus crispatus, Lactobacilluscurvatus, Lactobacillus delbrueckii, Lactobacillus desidiosus,Lactobacillus divergens, Lactobacillus enterii, Lactobacillusfarciminis, Lactobacillus fermentum, Lactobacillus frigidus,Lactobacillus fructivorans, Lactobacillus fructosus, Lactobacillusgasseri, Lactobacillus halotolerans, Lactobacillus helveticus,Lactobacillus heterohiochii, Lactobacillus hilgardii, Lactobacillushordniae, Lactobacillus inulinus, Lactobacillus jensenii, Lactobacillusjugurti, Lactobacillus kandleri, Lactobacillus kefir, Lactobacilluslactis, Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillusmalefermentans, Lactobacillus mali, Lactobacillus maltaromicus,Lactobacillus minor, Lactobacillus minutus, Lactobacillus mobilis,Lactobacillus murinus, Lactobacillus pentosus, Lactobacillus plantarum,Lactobacillus pseudoplantarum, Lactobacillus reuteri, Lactobacillusrhamnosus, Lactobacillus rogosae, Lactobacillus tolerans, Lactobacillustorquens, Lactobacillus ruminis, Lactobacillus sake, Lactobacillussalivarius, Lactobacillus sanfrancisco, Lactobacillus sharpeae,Lactobacillus trichodes, Lactobacillus vaccinostercus, Lactobacillusviridescens, Lactobacillus vitulinus, Lactobacillus xylosus,Lactobacillus yamanashiensis, Lactobacillus zeae, Pediococcusacidlactici, Pediococcus pentosaceus, Streptococcus cremoris,Streptococcus discetylactis, Streptococcus faecium, Streptococcusintermedius, Streptococcus lactis, Streptococcus thermophilus.

In one aspect of the invention, any of the above listed lactic acidproducing microorganisms may be used to inhibit or treat infections of ahost of pathogens, particularly bacterial pathogens including pathogenicbacteria such as Escherichia coli, Staphylococcus aureus, and Salmonellaspp., including Salmonella typhirium. These lactic acid producingmicroorganisms are particularly useful for inhibiting or treatinginfections of E. coli O157:H7. It has also been found that theseorganisms may be used for improving the performance of food animals byincreasing carcass weight, carcass quality, reducing carcass pathogens,and increasing average daily weight gain and feed efficiency ratio. Anyone of these microorganisms may be used for any of these purposes, orany combination of these microorganisms may be used.

In another aspect, the invention includes a formulation of a combinationof lactic acid producing microorganisms, such as those described in thepreceding paragraphs, with a second microorganism that enhances theeffectiveness of the lactic acid producing microorganisms in competingwith pathogenic microorganisms. Enhancing microorganisms that may beused in the formulations of the present invention are preferably lactateutilizing microorganisms. Examples of lactate utilizing microorganismsuseful in the present invention include but are not limited to:Megasphaerae eilsdenii, Peptostreptococcus asaccharolyticus,Propionibacterium freudenreichii, Propionibacterium acid-propionici,Propionibacterium freudenreichii, Propionibacterium globosum,Propionibacterium jensenii, Propionibacterium shermanii,Propionibacterium spp., and Selenomonas ruminantium. A therapeuticallyeffective amount of these enhancing microorganisms is a quantity thatproduces a beneficial therapeutic effect in the animal to which they areadministered, for example, a therapeutically effective amount of theseenhancing microorganisms may be greater than 1×10⁶ CFU/day, preferably1×10⁸ CFU/day, or even more preferably about 1×10⁹ CFU/day.

The use of specific microorganisms ensures that the desired effect isproduced locally. The different microorganisms used in the formulationshould be compatible with each other, for example, capable of growingtogether, and preferably, potentiating the other. Additionally, themicroorganisms preferably grow fast at the locus of action. Themicroorganisms may be selected for various characteristics, such asresistance to bile acids and/or commercial antibiotics, that make themmost suited for their intended use.

In a preferred mode, the formulations of the invention includeLactobacillus acidophilus, Lactobacillus crispatus, or Lactobacillusmurinus, either individually or in any combination. In another preferredmode, the formulations of the invention include Lactobacillusacidophilus, Lactobacillus crispatus, or Lactobacillus murinus, eitherindividually or in any combination with each other, and additionallyinclude Propionibacterium freudenreichii or Propionibacterium shermaniior both. Preferably, the formulations of the invention are applied tothe daily feed of beef or dairy cattle in a dry supplement, or in aliquid spray applied to the daily feed of the animals. The formulationsmay be administered once a day or over the course of a day, either inone meal, or divided among the meals, or in any other suitable way.

In Vitro Tests Examples 1 and 2

Several in vitro tests were conducted that demonstrate the ability ofparticular bacteria to effectively compete with and interfere with thegrowth of pathogenic bacteria such as E. coli O157:H7 and other.

Example 1

Lyophilized cultures of lactic acid producing and lactate utilizingorganisms were selected for their ability to inhibit the growth ofpathogens such as E. coli O157:H7, Streptococcus aureus and Salmonella.Combinations of the lactic acid producing and lactate utilizingorganisms were further selected for their ability to maximize theinhibition of growth of the various pathogens.

In order to identify those microorganisms that might be utilized in themethod and formulation of the invention, in vitro tests were conductedto identify particularly effective single strains. Seven strains ofPropionibacterium and six strains of Lactobacillus were screened fortheir ability to produce bacteriocins capable of creating zones ofinhibition on agar plates that were grown with E. coli O157:H7. Theresults of those tests are tabulated below.

TABLE 1 Inhibitory Activity of Propionibacterium Strains Grown inSelective Media PATHOGEN P9 P42 P79 P88 P93 P99 PF24 Gram + B. cereus NoNo No No No No No S. aureus Yes Yes Yes No No No No Gram − E. coliO157:H7 Yes Yes No No Yes Yes Yes Sal. typhirium No Yes No Yes Yes YesYes

TABLE 2 Inhibitory Activity of Lactobacillus Strains Grown in SelectiveMedia PATHOGEN 30SC 53545 381IL28 C28 FR3 R2 E. coli 43985 −4.1 16.891.8 89.7 88.7 64.9 O157:H7 E. coli 933 28.1 −3.4 92.7 93.5 91 89.4O157:H7 S. aureus 305 −15.6 −22.1 82.6 80.6 84.8 23.3

From the above tables, it may be appreciated that three strains ofLactobacillus lactic acid producing organisms—381IL28, C28 and FR3—andfour strains of Propionibacterium lactate utilizing organisms—P9, P42,P93 and P99—demonstrate the ability to inhibit the growth of pathogens,in particular, E. coli O157:H7. It should be recognized thatcombinations of lactic acid producing and lactate utilizingmicroorganisms can be selected for their ability to maximize theinhibition of growth of various pathogens.

Example 2

Selected strains of Lactobacillus acidophilus and Propionibacteriumfreudenreichii bacteria were grown in an in vitro comparison with E.coli on rich semi-anaerobic media at 38° C. to determine which strainscould effectively compete with E. coli growth under in vivo growthconditions. It was found that the LA51 and LA45 strains couldsubstantially out-grow the E. coli.

TABLE 3 Growth (Optical Density) of Selected Strains of Bacteria versusE. coli O157:H7 on Rich Semi-Anaerobic Media at 38° C. MINUTES E. coliO157:H7 LA45 LA51 PF24 0 0.2 0.2 0.2 0.2 50 0.3 0.38 0.55 0.3 90 0.450.65 0.84 0.35 120 0.60 0.85 1.0 0.36 200 0.80 1.2 1.28 0.38 230 0.851.25 1.28 0.39 365 0.90 1.25 1.28 0.50 440 0.90 1.25 1.28 0.58

In Vivo Tests Examples 3-9

In the following in vivo studies, ruminants were inoculated by providinga sufficient quantity of the bacterial strains tested along withnecessary growth medium components to the ruminants' intestines bynormal ingestion. Inhibited growth of pathogens such as E. coli O157:H7were observed in feedlot and dairy cattle, as well as other ruminantssuch as sheep, goats and game. Various inoculation processes wereutilized. Examples of these inoculation processes include:

-   -   Placing lyophilized cultures in water, and then spraying or        blending the mixture onto the feed of the animal. The mixture        can be in dry form, together with additional carriers that are        added to the diet of the animal. The diet can include one or        more ingredients such as corn, cereal grains, corn byproducts,        cereal grain byproducts, alfalfa hay, corn silage, small grain        silage, grass hay, plant stalks, oilseed byproducts, protein        meals, urea, minerals, molasses, and various fat and oil        products.    -   Suspending lyophilized cultures in various oils, water and/or        compounds for providing a drench to be supplied directly to the        animal and the digestive tract of the animal.    -   Adding the lyophilized cultures to the drinking water of the        animals.

Example 3

In vivo tests were conducted with combinations of lactic acid producingand lactate utilizing microorganisms for the inhibition of the pathogenE. coli OP157:H7. The results of those tests are tabulated below inTables 4 and 5.

TABLE 4 Inhibition of E. coli O157:H7 in Manure at 37° C. TREATMENT 0Hours 24 Hours Control 5.74 6.56 Combination PF24, LA45, LA51 5.74 4.48

TABLE 5 Inhibition of E. coli O157:H7 in Rumen Fluid at 37° C. TREATMENT0 Hours 24 Hours 48 Hours Control 6.64 6.70 6.79 Combination PF24, LA45,LA51 5.56 5.04 5.00

The data is reported at log₁₀ CFU E. coli O157:H7/ml. The organismsPF24, LA45 and LA51 are all able to function at a pH of about 4.0 toabout 5.0 up to above a pH of about 7.0. In beef production, the chiefconcern is inhibiting pathogens of cattle on a finishing diet containinghigh levels of concentrate that tend to decrease rumen pH from about 7.0to the range of about 5.0 to about 6.9, a range in which the formulationof the present invention preferably functions. While the above in vivotests illustrate the use of the lactate utilizing organism PF24 and thelactic acid producing organisms LA45 and LA51, it should be understoodthat the present invention is not limited to these organisms as thereare many strains that can be adapted for the formulation and method ofthe invention.

In vivo testing has also been conducted with single strains forassessing their effectiveness of pathogen growth inhibition, includingE. coli O157:H7. In particular, M35 and LA51 each demonstrate theability to inhibit shedding of E. coli O157:H7 by about fifty percentabove the level of the control animals.

Example 4

One hundred and eighty (180) cattle were sorted by weight and put intopens of five (5) animals per pen. During weighing, a fecal sample wastaken directly from the rectum of each animal. Initially, only 3 of the180 animals tested positive for E. coli O157:H7. The animals weremonitored for shedding on a bi-weekly basis by taking a composite samplefrom five fresh droppings on the floor of each pen. Two weeks after thesorting period, twenty-five (25) of the thirty-six (36) pens, or 69%,were positive for E. coli O157:H7. Four weeks after sorting, theprevalence had declined to seven pens that were positive, or 19.4%.

With approximately sixty days left in the feeding period, the cattlewere weighed, resorted, and individual animals were again tested forshedding of the pathogen. A total of twenty-six (26) animals, or 14.4%,were shedding the pathogen. The animals were re-sorted based on weightand shedding pattern. Animal treatment began at this time.

Two separate treatments utilizing two separate types of lactic acidproducing bacteria (NPC 747 and NPC 750) were administered to test theirability to reduce E. coli O157:H7 in the study animals.

Pen tests taken one week following the beginning of the treatment periodindicated that 25% of the pens receiving no treatment were positive forE. coli O157:H7, while 8% of the pens receiving NPC 750 treatment werepositive and 0% of the pens receiving NPC 747 treatment were positive.Two weeks after the treatments, 50% of the samples taken from thecontrol (untreated) animals were positive, while only 30% and 20% of thesamples from animals receiving NPC 750 and NPC 747 treatments,respectively, were positive for E. coli O157:H7. The tests indicate areduction in the shedding rate by approximately one-half that of thecontrol for those receiving NPC 747 treatment, which was a greaterreduction than for those receiving NPC 750 treatment. All animalsshedding E. coli O157:H7 prior to receiving NPC 747 treatment testednegative after receiving the treatment. Furthermore, the pathogen didnot spread to other animals in the same pen. The majority of the controlanimals that tested positive at the beginning of the administration ofthe treatment continued to test positive, with other animals in the samepen beginning to shed the pathogen.

On day 42, there were significant (P<0.05) differences among treatmentsfor the individual animal samples. Ten percent (10%) of the animals fedthe NPC747 strain were positive, whereas 20% of the animals fed NPC 750were positive. In contrast, 58% of the control animals were positive.

The animals were sampled pre-slaughter. The animals receiving NPC 747treatment had significantly (P<0.05) less detectable E. coli O157:H7with only 3.3% of the animals testing positive. The animals receivingthe NPC750 strain and those in the control group were not significantlydifferent, with 15% and 20% shedding, respectively.

Fecal samples taken in the slaughter plant indicated that 3.3% of theNPC 747 treated animals were positive, 6.6% of the NPC 750 treatedanimals were positive, and 10% of the control animals were positive.Averaging all samples over all sampling times, 61.7% of the controlanimals shed the pathogen during the feeding period, 51.7% of the NPC750 animals shed the pathogen, and only 35% of the NPC 747 treatedanimals shed the pathogen.

Example 5

One hundred (100) steers were placed into pens of ten (10) animals perpen. Initially, a fecal sample was taken directly from the rectum of twosteers per pen. Another sample was taken 170 days later. The sameanimals were sampled during both sampling times. A control groupreceiving no treatment was included in the study. The animals that weretreated were treated with the Lactobacillus acidophilus strain LA51(purchased under the trade name NPC2000). All groups were fed Rumensinand Tylan. The results of the assays are tabulated below.

TABLE 6 Inhibition of E. coli Treated with NPC 2000 Initial Second FinalTREATMENT E. coli + % + E. coli + % + E. coli + % + Control 2 of 20 10 4of 20 20 3 of 20 15 LA51 5 of 20 25 0 of 20 0 0 of 20 0

TABLE 7 Animal Performance Data After 198 Days (Final Results) InitialLive Final Live Hot Carcass Average Daily Weight, Weight, Weight, WeightGain, TREATMENT lbs. lbs. lbs. lbs. Control 772 1560 945 3.98 LA 51 7721626 984 4.31 Response, lb. 66 39 0.33 % Response 4.23 4.12 8.30

Example 6

A study was conducted to determine whether food-grade probiotic bacteriacould reduce fecal shedding of E. coli O157:H7 in experimentallyinfected weaned beef calves. The probiotic bacteria in the studyincluded various bovine fecal Lactobacillus spp. isolates that wereselected on the basis of high-level in vitro toxic activity against E.coli O157:H7.

Five 7-month old beef calves were subjected to rumen cannulation surgeryand, following recovery, housed in biosafety level 3 isolation rooms.The calves were inoculated intra-ruminally once daily for a sixty-dayperiod with 1×10⁹ CFU of one of the probiotic bacterial strains listedin Table 8 below.

Two weeks after initiation of probiotic administration, the calves werechallenged by intra-ruminal inoculation with E. coli O157:H7 (C1).Fifteen (C2) and twenty seven (C3) days after the first inoculation(C1), the calves were again challenged. The first inoculation C1comprised a total of about 1×10⁹ CFU of a combination of strains 920,922, 944 and 966. The second inoculation C2 was with a total of about1.63×10¹¹ CFU of these strains. The third inoculation C3 included about1×10⁹ CFU of strain 86-24.

Prior to and after each challenge, the calves were tested daily forfecal shedding of the inoculum strains. Every two weeks, the animalswere tested for evidence of immunity as assessed by serum antibodytiters to the Tir protein and 0157 lipopolysaccharide (LPS) antigen. Theresults are shown in Table 8 below. All calves prior to challenge had arelatively high anti-Tir antibody titer that provided the calves with asignificant level of immunity against the challenge strains, as allcalves, including the control, had a S:C ratio of less than onefollowing the first C1 and second C2 treatments.

TABLE 8 Comparison of the Effect of Feeding Different Lactobacillus spp.Probiotic Strains on E. coli O157:H7 Shedding in Weaned Beef CalvesProbiotic strain and time of Antibody Titers No. Days TotalShedding:Challenge challenge Tir O157 of Shedding Shedding Ratio M35 C11:12,800 1:12,800 3 1.45 × 10⁶ (S1) 0.00145 (S1/C1) C2 1:12,800 1:12,8002 3.59 × 10⁶ (S2) 0.000022 (S2/C2) C3 1:12,800 1:12,800 3 3.14 × 10⁸(S3) 0.314469 (S3/C3) Total 1:51,200 1:51,200 8 3.2 × 10⁸ (S) 0.001936(S/C) LA45 C1 1:6,400 1:12,800 3 1.8 × 10⁶ (S1) 0.0017955 (S1/C1) C21:6,400 1:12,800 2 5.13 × 10⁶ (S2) 0.0000031 (S2/C2) C3 1:6,400 1:12,80019 1.84 × 10¹¹ (S3) 184.4 (S3/C3) Total 1:12,800 1:51,200 24 1.84 × 10¹¹(S) 1.1759 (S/C) PBS C1 1:25,600 1:12,800 2 1.0 × 10⁴ (S1) 0.001 (S1/C1)C2 1:25,600 1:12,800 1 1.47 × 10⁹ (S2) 0.009 (S2/C2) C3 1:25,6001:12,800 8 2.07 × 10⁸ (S3) 0.207 (S3/C3) Total ND 1:51,200 11 1.87 × 10⁹(S) 0.01 (S/C) LA51 C1 1:6,400 1:12,800 2 9.41 × 10⁶ (S1) 0.0009405(S1/C1) C2 1:6,400 1:12,800 1 5.13 × 10⁶ (S2) 0.0000031 (S2/C2) C3 ND1:12,800 9 2.62 × 10⁸ (S3) 0.26163 (S3/C3) Total 1:6,400 1:51,200 122.63 × 10⁸ (S) 0.0015944 (S/C) L411 C1 1:12,800 1:6,400 2 1.03 × 10⁴(S1) 0.001026 (S1/C1) C2 1:12,800 1:6,400 8 6.44 × 10⁸ (S2) 0.0039529(S2/C2) C3 1:12,800 1:6,400 4 5.13 × 10⁹ (S3) 0.0513 (S3/C3) Total1:51,200 1:25,600 14 6.97 × 10⁹ (S) 0.0042221 (S/C)

The shedding/challenge ratio in the above table represents the totalamount of E. coli O157:H7 shed after inoculation. This number normalizesthe values allowing more accurate comparison of the animals andproviding more meaningful information than just reviewing the totalnumber of days the cattle shed the organism. M35, LA45, LA51 and L411represent the various Lactobacillus strains tested. PBS represents thecontrol animal. The total shedding is the CFU per gram in feces timesthe fecal output in grams on a positive day of shedding times the totalnumber of positive days of shedding.

Because the anti-Tir titers were not significantly different among thecalves, three of the four probiotics have an effect based upon thefollowing reduction in the S:C ratio compared to the control—80% for thecalf feed M35, 84% for the calf fed LA51, and 58% for the calf feed 411.Further, the animals fed M35 experienced a 27% reduction in the numberof days of shedding compared to the control J3. However, in the animalfed LA51, there was a 9% increase in the number of days of sheddingcompared to the control J3. Accordingly, feeding probiotics is effectivein the reduction of E. coli O157:H7 fecal shedding in cattle.

Example 7

A study was conducted to determine whether combinations of lactateutilizing and lactic acid producing bacteria added to the feed of dairycows could reduce pathogens in and improve the milk production of dairycows. Three sets of dairy cows were tested. The first set of dairy cowswas the control group (Group 1). The second set of dairy cows wereadministered the lactate utilizing bacterium Propionibacteriumfreudenreichii strain PF24 in combination with the lactic acid producingbacterium Lactobacillus acidophilus strain NPC 747 in accordance withthe methods set forth in the preceding sections (Group 2). The third setof dairy cows were administered the lactate utilizing bacteriumPropionibacterium freudenreichii strain PF24 in combination with twostains of the lactic acid producing bacterium Lactobaciullusacidophilus, LA51 (NPC747) and LA45, in accordance with the methods setforth in the preceding sections (Group 3). The results of this study areset forth in table 9.

Table 9 demonstrates the effects of each of the treatment regimes on themilk production, body weight, and feed consumption of the dairy cows.The data demonstrates that treatments involving feeding the dairy cowslactate utilizing bacteria in combination with lactic acid producingbacteria resulted in statistically significant improvements in thequantity of milk produced, the quantity of fat corrected milk produced(i.e., milk with a higher fat content is weighted more), the ratio offat corrected milk produced per quantity of feed consumed, the quantityof energy corrected milk produced (i.e., milk with a higher caloriecontent is weighted more), the quantity of energy corrected milkproduced per quantity of feed consumed, the quantity of milk fat in themilk produced, and the urea content of the cows' blood serum. Theaddition of the LA45 strain in Group 3 resulted in an increase in theurea content of the cows' blood serum.

TABLE 9 The Effect of Addition of Bacterial Cultures on PerformanceVariables for Lactating Dairy Cows Treatment Contrasts Treatments (P <0.10) Group Group Group Standard Group 1 vs. Group 2 vs. Variable 1 2 3Error Groups 2 and 3 Group 3 DMI², kg/day 25.5 26.2 26.3 0.5  NS¹ NSMilk, kg/day 37.7 39.6 38.5 0.7 0.08 NS FCM³, kg/day 34.9 37.8 37.6 0.8 0.007 NS Milk/DMI, kg/kg 1.45 1.53 1.48 0.04 NS NS FCM/DMI, kg/kg 1.351.45 1.44 0.03 0.03 NS ECM⁴, kg/day 34.5 36.8 37.0 0.8 0.03 NS ECM/DMI,kg/kg 1.34 1.42 1.41 0.04 0.03 NS Milk fat, % 3.19 3.23 3.40 0.10 NS NSMilk fat, kg/day 1.15 1.27 1.29 0.04 0.02 NS Milk protein, % 2.95 2.943.00 0.05 NS NS Milk protein, kg/day 1.09 1.13 1.12 0.03 NS NS Somaticcells/ml, ×1000 259 198 257 111 NS NS Final Body Weight, kg 667.6 658.3664.1 6.5 NS NS Body Weight Change, kg 28.9 20.8 21.2 7.3 NS NS Serumurea N, mg/dl 22.62 20.43 21.66 0.48 0.01 0.08 Serum glucose, mg/dl64.73 67.55 65.52 1.19 NS NS ¹Not statistically significant for P > 0.10²Dry matter intake ³3.5% fat-corrected milk ⁴Energy-corrected milk forcomparison on an equivalent calorie basis

Table 10 shows a marked reduction in the occurrence of the pathogen E.coli O157:H7 in fecal samples from each of the sets of dairy cows towhich the combination of lactate utilizing bacteria and lactic acidproducing bacteria were administered. The effect was particularlypronounced in the dairy cows administered both the LA51 (NPC747) andLA45 strains of L. acidophilus in combination with the PF24 strain of P.freudenreichii. In this set of cows, no E. coli O157:H7 was detected inany fecal samples.

TABLE 10 Occurrence of E. coli O157:H7 in Fecal Samples from Dairy CowsGroup 1 Group 2 Group 3 E. coli O157:H7 19% 12% 0% occurrence

Example 8

A study was conducted to determine whether particular novel combinationsof bacteria could reduce the occurrence of pathogenic bacteria. It wasalso found that these combinations improved the feeding efficiency ofcattle. Two hundred forty (240) steers were assigned to 48 pens of fivehead each. The average weight of the steers was 780 lbs. Each pen wasallotted one of four treatments: (1) group 1 was the control group andwas fed no microorganisms, (2) group 2 was fed two strains:Propionibacterium freudenreichii strain PF24 and Lactobacillusacidophilus strain LA51 (NPC747), each strain in an amount of 1×10⁹CFU/day, (3) group 3 was fed three strains: PF24 in an amount of 1×10⁹CFU/day, LA51 (NPC747) in an amount of 1×10⁹ CFU/day, and Lactobacillusacidophilus strain LA45 in an amount of 1×10⁶ CFU/day, and (4) group 4was fed three strains: PF24 in an amount of 1×10⁹ CFU/day, LA51 (NPC747)in an amount of 1×10⁶ CFU/day, and Lactobacillus acidophilus strain LA45in an amount of 1×10⁶ CFU/day.

Table 11 demonstrates an improvement in the feeding efficiencies of thegroups administered the novel combinations of bacteria. All of thegroups administered the novel combinations of bacteria exhibited agreater average daily weight gain over the course of 56 and 140 daysrelative to the control group.

TABLE 11 Feed Efficiencies Average Over Days 0-56 Average Over Days0-140 Average Feed Average Feed Daily Gain Intake Daily Gain IntakeGroup 1 (control) 4.42 lbs. 19.16 lbs. 3.62 lbs. 18.82 lbs. Group 2 4.52lbs. 19.62 lbs. 3.70 lbs. 19.32 lbs. Group 3 4.54 lbs. 19.24 lbs. 3.72lbs. 18.79 lbs. Group 4 4.61 lbs. 19.61 lbs. 3.69 lbs. 19.31 lbs.

Table 12 demonstrates a substantial improvement in the quantity of E.coli O157:H7 found in the carcasses and hides of the steers followingslaughter. Notably, the carcasses of the steers in Group 2 exhibitedless than half of this pathogen than the control group, and thecarcasses of the steers in the other two groups also exhibited asubstantial reduction in the quantity of this pathogen. Particularlynotable is the dramatic reduction in the quantity of E. coli in thehides of all of the steers who were administered the formulations of theinvention.

TABLE 12 E. coli O157:H7 incidence Carcass Hide Group 1 (control) 33.3%20%  Group 2 13.3% 0% Group 3 26.6% 0% Group 4  20% 0%

Example 9

A study was conducted to determine which of several methods ofcontrolling pathogenic growth in cattle was superior. The first methodinvolved feeding the bacteria NPC 747 and NPC 750 (also known as M35,available under that name from the University of Nebraska) to cattle.The second method involved removing starch from the cattle's diet. Thethird method involved pen cleaning. The study design was 3 H 2 H 2factorial. A finishing diet (33% high moisture corn, 20% dry rolledcorn, 40% wet corn gluten feed, and 7% alfalfa, with vitamins, minerals,Rumensin, and Tylan) was fed to 432 steers (average weight 340 kg) in 54pens, with 8 steers in each pen. The bacteria NPC 747 and NPC 750 wasfed daily to the cattle in 18 pens. Half the pens were cleaned monthly,and the other half were cleaned only at the end of the study. Two weeksprior to slaughter, the diet was changed for half the cattle, with cornbran replacing corn in the cattle's feed.

Neither the first nor third methods affected steer performance (P>0.39),but diet change reduced DMI (P<0.001; 12.8 kg/d versus 11.5 kg/d) duringthe last two weeks, and reduced ADG and efficiency for the entirefeeding period (P<0.001). Carcass weight was reduced 8.4 kg by dietchange.

Individual fecal samples were obtained monthly and 0, 1, and 2 weeksprior to slaughter and analyzed for E. coli O157:H7. An entire pen wasthe experimental detection unit, and a pen was deemed positive for E.coli O157:H7 if any of the 8 steers was positive. Overall detection ofE. coli O157:H7 was low (145/3024 animal-weeks). The second and thirdmethods had no effect on E. coli O157:H7 prevalence. The first methodnumerically reduced E. coli 0157 positive pens the week of marketing(44% versus 17%; P=0.10).

While various embodiments of the invention have been described above, itshould be understood that they have been presented by way of exampleonly, and not limitation. Thus, the breadth and scope of the presentinvention should not be limited by any of the above-described exemplaryembodiments, but should be defined only in accordance with the followingclaims and their equivalents.

What is claimed is:
 1. A method of inhibiting an intestinal pathogenicinfection in a ruminant, the method comprising: (a) administering to theruminant a composition that inhibits the growth of the pathogenicbacterium in the ruminant, said composition comprising a lactic acidproducing bacterium and a lactate utilizing bacterium, wherein the ratioin colony forming unit (CFU) between said lactic acid producingbacterium and said lactate utilizing bacterium in said composition isabout 1:1 (CFU_(lactic acid producing bacterium):CFU_(lactate utilizing bacterium)), and (b) assessing the effects ofpathogen reduction by said composition, said lactic acid producingbacterium being the LA51 strain, said lactate utilizing bacterium beingthe PF24 strain, wherein said LA51 and PF24 strains are eachadministered to said ruminant at 1×10⁹ CFU/day.
 2. The method of claim1, wherein the pathogenic bacterium is selected from the groupconsisting of Escherichia coli, Salmonella spp., and Staphylococcusaureus.
 3. The method of claim 2, wherein the pathogenic bacterium isEscherichia coli.
 4. The method of claim 3, wherein the Escherichia coliis Escherichia coli O157:H7.